Glucose dehydrogenase

ABSTRACT

Disclosed is a modified glucose dehydrogenase having pyrroloquinoline quinone as a coenzyme, wherein one or more amino acid residues in a region of 186-206 amino acid of water-soluble PQQGDH derived from  Acinetobacter calcoaceticus  or in an equivalent region from other species are replaced with other amino acid residues. Also disclosed is a gene coding for the modified glucose dehydrogenase of the invention, a vector comprising the gene of the invention and a transformant comprising the vector, as well as a glucose assay kit and a glucose sensor comprising the modified glucose dehydrogenase of the invention.

TECHNICAL FIELD

The present invention relates to a glucose dehydrogenase havingpyrroloquinoline quinone as a coenzyme (PQQGDH), and its preparation andapplication to glucose quantification.

BACKGROUND OF THE INVENTION

Blood glucose concentration is a important marker for diabetesdiagnosis. In addition, quantification of glucose concentration is usedin monitoring the process of fermentative production usingmicroorganisms. Conventionally, glucose quantification is performed byan enzymatic method using glucose oxidase (GOD) or glucose-6-phosphatedehydrogenase (G6PDH). However, the GOD method requires addition ofcatalase or peroxidase into the assay system to quantify hydrogenperoxide levels generated by oxidative reaction of glucose. G6PDH hasbeen used for glucose quantification based on spectroscopy. This methodinvolves the addition of coenzyme NAD(P) into the assay system.

Recently the application of PQQGDH, an enzyme which usespyrroloquinoline quinine as a coenzyme is attracting attention, in placeof the enzyme used in the existing glucose quantification method. PQQGDHis a glucose dehydrogenase having pyrroloquinoline quinone as acoenzyme, and catalyzes the reaction of oxidizing glucose to producegluconolactone.

Two types of PQQGDHs are known: membrane-bound and water-soluble.Membrane-bound PQQGDH is a single-peptide protein with an approximatemolecular weight of 87 kDa, and is found in a wide variety ofgram-negative bacteria. See, for example, J. Bacteriol. (1990) 172,6308-6315, A. M. Cleton-Jansen et al. On the other hand, water-solublePQQGDH has been found in some strains of Acinetobacter calcoaceticus(Biosci. Biotech. Biochem. (1995), 59 (8), 1548-1555), and itsstructural gene has been cloned and its amino acid sequence determined(Mol. Gen. Genet. (1989), 217:430-436). Water-soluble PQQGDH derivedfrom A. calcoaceticus is a homodimer enzyme with an approximatemolecular weight of 50 kDa. It shows little homology in primarystructure with other PQQ enzymes.

Recently, the results of X-ray structural analysis of water-solublePQQGDH were published and its conformation and active center wererevealed (A. Oubrie, et al. (1999) J. Mol. Bio., 289, 319-333, A.Oubrie, et al. (1999) The EMBO Journal, 18 (19), 5187-5194, and A.Oubrie, et al. (1999), PNAS 96 (21), 11787-11791). These reportsdemonstrate that water-soluble PQQGDH is a β-propeller proteinconsisting of six W-motifs.

PQQGDH is expected to have potential in glucose assays, for example, asa recognition device of a glucose sensor, because it has highlyoxidative activity towards glucose and does not require oxygen as anelectron acceptor as it is complexed with a coenzyme. However, the lowselectivity of PQQGDH for glucose was an obstacle to this use.

The object of this invention is to provide a modified water-solublePQQGDH with a high selectivity for glucose.

DISCLOSURE OF THE INVENTION

As a result of extensive research to engineer conventional water-solublePQQGDH to develop a PQQGDH which shows a higher selectivity for glucoseand can be applied to clinical diagnosis and food analysis, the inventorsuccessfully obtained an enzyme with higher selectivity by introducingamino acid mutations at certain regions of water-soluble PQQGDH.

The present invention provides a modified water-soluble glucosedehydrogenase having pyrroloquinoline quinone as a coenzyme, wherein oneor more amino acid residues of a wild type water-soluble glucosedehydrogenase are replaced with other amino acid residues and havinghigh selectivity for glucose compared with the wild type water-solubleglucose dehydrogenase. The modified glucose dehydrogenase of theinvention has higher glucose selectivity compared to the wild typewater-soluble glucose dehydrogenase. Preferably, the modified glucosedehydrogenase of the invention has lower reactivity to lactose andmaltose than to glucose compared to the wild type enzyme. Morepreferably, the modified glucose dehydrogenase of the invention hasreactivity to lactose or maltose of less than 50%, even more preferably40%, and most preferably 30% of the reactivity to glucose (100%).

One aspect of the invention provides a modified glucose dehydrogenasewherein one or more amino acid residues in a region of 186-206 aminoacid of water-soluble PQQGDH derived from Acinetobacter calcoaceticus orin an equivalent region from other species are replaced with other aminoacid residues (i.e., amino acid residues different from those found in anaturally occurring PQQ glucose dehydrogenase). Note that the amino acidnumbering in this specification starts from the initiator methionine asthe +1 position.

As used herein, the term “equivalent” with reference to the positions orregions of amino acid residues means that some amino acid residues orregions have an equivalent biological or biochemical function in two ormore proteins which are structurally similar but not identical. Forexample, a certain region in water-soluble PQQGDH derived from organismsother than Acinetobacter calcoaceticus is said to be “equivalent to theregion of amino acid residues 162-182 of water-soluble PQQGDH derivedfrom Acinetobacter calcoaceticus” when the amino acid sequence of such aregion has a high similarity to the amino acid sequence in the 162-182region of water-soluble PQQGDH derived from Acinetobacter calcoaceticus,and the same function can be reasonably predicted based on the secondarystructure of the relevant regions in the proteins. Additionally, the 7thamino acid residue of that region is said to be “an amino acid residueat the equivalent position to the amino acid residue 168 ofwater-soluble PQQGDH derived from Acinetobacter calcoaceticus”.

Preferably, in the modified glucose dehydrogenase of the invention,glutamine at position 168 or leucine at position 169 of the amino acidse uence defined in SEQ ID NO: 1 of water-soluble PQQGDH derived fromAcinetobacter calcoaceticus or an amino acid residue in an equivalentposition from other species are replaced with another amino acidresidues.

In another aspect, the invention features a modified glucosedehydrogenase having pyrroloquinoline quinone as a coenzyme whereinglutamine at position 168 of the amino acid sequence defined in SEQ IDNO: 1 is replaced with another amino acid residue. Preferably, glutamineat position 168 of the amino acid sequence defined in SEQ ID NO: 1 isreplaced with alanine, glycine, glutamic acid, leucine, phenylalanine,serine or aspartic acid.

In another aspect, the invention features a modified glucosedehydrogenase having pyrroloquinoline quinone as a coenzyme wherein bothglutamine at position 168 and aspartate at position 143 of the aminoacid sequence defined in SEQ ID NO: 1 are repliaced with other aminoacid residues. Preferably, glutamine at position 168 of the ammo acidsequence defined in SEQ ID NO: 1 is replaced with alanine, glycine,glutamic acid, leucine, phenylalanine, seine or aspartic acid. Morepreferably, aspartate at position 143 of the amino acid sequence definedin SEQ ID NO: 1 is replaced with glutamic acid, and glutamine atposition 168 is replaced with alanine, glycine, glutamic acid, leucine,phenylalanine, seine or aspartic acid.

In another aspect, the invention features a modified glucosedehydrogenase having pyrroloquinoline quinone as a coenzyme whereinaspartate at position 143 of the amino acid sequence defined in SEQ IDNO: 1 is replaced with another amino acid residue, and asparagine atposition 428 is replaced with another amino acid residue. Preferably,spartate at position 143 of the amino acid sequence defined in SEQ IDNO: 1 is replaced with glutamic acid. More preferably, aspartate atposition 143 of the amino acid sequence defined in SEQ ID NO: 1 isreplaced wit glutamic acid, and asparagine at position 428 is replacedwith threonine.

In another aspect, the invention features a modified glucosedehydrogenase having pyrroloquinoline quinone as a coenzyme whereinglutamine at position 168 of the amino acid sequence defined in SEQ IDNO: 1 is replaced with another amino acid residue, and asparagine atposition 428 is replaced with another amino acid residue. Preferably,glutamine at position 168 of the amino acid sequence defined in SEQ IDNO: 1 is replaced with alanine, glycine, glutamic acid, leucine,phenylalanine, seine or aspartic acid, and asparagine at position 428 isreplaced wit another amino acid residue. More preferably, glutamine atposition 168 of the amino acid sequence defined in SEQ ID NO: 1 isreplaced with alanine, glycine, glutamic acid, leucine, phenylalanine,serine or aspartic acid, and asparagine at position 428 is replaced withthreonine.

In another aspect, the invention features a modified glucosedehydrogenase having pyrroloquinoline quinone as a coenzyme whereinleucine at position 169 of the amino acid sequence defined in SEQ ID NO:1 is replaced with another amino acid residue. Preferably, leucine atposition 169 of the antno acid sequence defined in SEQ ID NO: 1 isreplaced with alanine, glycine, methionine, tryptophan or lysine.

In another aspect, the modified glucose dehydrogenase of the inventioncomprises the amino acid sequence:

Gly-Arg-Asn-Xaa1-Xaa2-Ala-Tyr-Leu (SEQ ID NO: 3)wherein Xaa1 and Xaa2 are independently any amino acid residues,provided that when Xaa1 is Gln, then Xaa2 is not Leu. Preferably, Xaa1is Ala, Gly, Glu, Leu, Phe, Ser or Asp, and Xaa2 is Ala or Gly.

The invention also provides a gene coding for the modified glucosedehydrogenase of the invention, a vector comprising the gene of theinvention and a transformant comprising the vector, as well as a glucoseassay kit and a glucose sensor comprising the modified glucosedehydrogenase of the invention.

Since the enzyme protein of the modified glucose dehydrogenase of theinvention shows high selectivity and high oxidization activity toglucose, it can be applied to highly specific and sensitive measurementof glucose.

BRIEF EXPLANATIONS OF DRAWINGS

FIG. 1 shows a structure of pGB2 plasmid used to construct mutant genesencoding modified PQQGDHs of the present invention.

FIG. 2 shows a method of constructing mutant genes encoding modifiedPQQGDHs of the present invention.

FIG. 3 is a graph showing substrate concentration dependency of theactivity of the modified PQQGDHs of the present invention.

DETAILED EXPLANATIONS OF THE INVENTION

Structure of Modified PQQGDH

In a preferred modified glucose dehydrogenase of the invention, one ormore amino acid residues in the 162-182 amino acid region ofwater-soluble PQQGDH derived from Acinetobacter calcoaceticus or in anequivalent region from other species are replaced with other amino acidresidues. Preferably, glutamine at position 168 of the amino acidsequence defined in SEQ ID NO: 1 is replaced with alanine or glycine,and/or leucine at position 169 is replaced with alanine, glycine,methionine, tryptophan or lysine.

In another aspect of the modified PQQGDH of the invention, in additionto the modifications as described above, aspartate at position 143 ofthe amino acid sequence defined in SEQ ID NO: 1 is also replaced withanother amino acid, preferably with glutamic acid. Also preferably, inthe modified PQQGDH of the present invention, in addition to themodifications as described above, asparagine at position 428 of theamino acid sequence defined in SEQ ID NO: 1 is also replaced withanother amino acid, preferably with threonine. Involvement of aspartateat position 143 and asparagine at position 428 of the amino acidsequence defined in SEQ ID NO: 1 in recognition and binding of substrateby PQQGDH is described in Japanese Patent Public Disclosure Nos.2001-346587 and 2001-197888, respectively. In general, however, noprediction can be made regarding the changes of substrate selectivityand enzyme activity which may be caused by simultaneously altering theamino acid residues in different domains. In some cases the enzymeactivity will be completely abolished. Therefore, it was a surprisingdiscovery in the present invention that improved selectivity for glucosecan be achieved by introducing double mutations.

In another aspect, the modified glucose dehydrogenase of the presentinvention comprises the amino acid sequence:Gly-Arg-Asn-Xaa1-Xaa2-Ala-Tyr-Leu (SEQ ID NO: 3) wherein Xaa1 and Xaa2are independently any amino acid residues, provided that when Xaa1 isGln, then Xaa2 is not Leu. Preferably, Xaa1 is Ala, Gly, Glu, Leu, Phe,Ser or Asn, and Xaa2 is Ala or Gly.

Preparation Method of Modified PQQGDH

The sequence of the gene encoding wild type water-soluble PQQGDH derivedfrom Acinetobacter calcoaceticus is defined in SEQ ID NO:2. Genesencoding modified PQQGDHs of the present invention can be constructed byreplacing the nucleotide sequences encoding certain amino acids of thewild type water-soluble PQQGDH with the nucleotide sequences encodingthe amino acids to be replaced. A wide range of methods forsite-specific mutagenesis have been elaborated in the art, as describedin, for example, Sambrook et al., “Molecular cloning; A LaboratoryManual”, second edition, 1989, Cold Spring Harbor Laboratory Press, NewYork.

The mutant gene obtained in this manner is inserted into an expressionvector (such as a plasmid) and transformed into an appropriate host(such as E. coli). A wide variety of host-vector systems have beendeveloped in the art to express exogenous proteins. For example,bacteria, yeast, and cultured cells can be used as hosts.

As long as its glucose dehydrogenase activity is retained, the modifiedPQQGDH of the invention can further contain deletion, substitution oraddition of other amino acid residues. A wide range of methods forsite-specific substitution are available in the art.

Moreover, those skilled in the art can determine a region in awater-soluble PQQGDH derived from other bacteria which is equivalent tothe region of the amino acid residues 162-182 of the water-solublePQQGDH derived from Acinetobacter calcoaceticus by comparing the arrayof the primary structure of the proteins, or by comparing the secondarystructures predicted from the primary structures of the enzymes. Thus,additional modified glucose dehydrogenases with improved glucoseselectivity can be obtained by substituting amino acid residues in thisregion with another amino acid residues. Such modified glucosedehydrogenases are also within the scope of the present invention.

After culturing the transformants expressing modified PQQGDH, obtainedas described above, the cells are collected by centrifugation and thencrushed by French press, or the periplasmic enzymes may be released intothe medium by osmotic shock. After ultracentrifugation, water solublefractions containing PQQGDH can be obtained. Alternatively, expressedPQQGDH can be secreted into the culture by using an appropriatehost-vector system. The water soluble fraction thus obtained is thenpurified by ion-exchange chromatography, affinity chromatography orHPLC, to obtain the modified PQQGDH of the invention.

Measurement of Enzyme Activity

The PQQGDH of the present invention catalyzes oxidation of glucose toproduce gluconolactone using PQQ as a coenzyme. The enzyme activity canbe quantified by color-developing reaction of a redox dye to measure theamount of PQQ reduced with glucose oxidation by PQQGDH. Example ofcolor-developing reagents include PMS (Phenazine methosulfate), DCIP(2,6-dichlorophenolindophenol), potassium ferricyanide, and ferrocene.

Glucose Selectivity

The glucose selectivity of the present invention can be evaluated bymeasuring relative enzyme activity with respect to the activity forglucose by using a variety of sugars such as 2-deoxy-D-glucose, mannose,allose, 3-o-methyl-D-glucose, galactose, xylose, lactose, and maltose asa substrate.

The modified PQQGDH of the present invention shows improved glucoseselectivity compared with wild type enzyme. Especially it has highreactivity to glucose compared with that to maltose. Therefore, theassay kit and enzyme sensor prepared using the modified enzyme of theinvention will exhibit high selectivity for glucose, and will haveadvantages in detecting glucose with high sensitivity even in samplescontaining variety of sugars.

Glucose Assay Kit

The present invention also provides a glucose assay kit containing themodified PQQGDH of the invention. The glucose assay kit of the inventionmay contain a sufficient quantity of the modified PQQGDH to carry out atleast one assay. Besides modified PQQGDH, the kit may typically comprisebuffers required for assay, a mediator, a standard solution of glucoseto generate a calibration curve, and instructions for use. The modifiedPQQGDH can be supplied in a variety of forms, for example, asfreeze-dried reagent or appropriate stock solutions. Preferably, themodified PQQGDH of the present invention may be supplied in the form ofa holoenzyme, but can be supplied in the form of apoenzyme and convertedinto a holoenzyme before use.

Glucose Sensor

The present invention also provides a glucose sensor containing themodified PQQGDH of the invention. Carbon, gold, or platinum may be usedas an electrode, and the enzyme of the present invention is immobilizedon the electrode. Immobilization methods includes, for example, methodsusing cross-linking reagents, inclusion into a macromolecular matrix,coating with dialysis membrane, methods using photo-crosslinkingpolymer, electric conductive polymer, and redox polymer. The enzyme canalso be immobilized in a polymer or adsorbed on the electrode togetherwith an electron mediator, such as ferrocene or its derivative.Combinations of the above may also be used. Preferably, the modifiedPQQGDH of the present invention is immobilized on the electrode in theform of a holoenzyme, but can also be immobilized in the form ofapoenzyme and PQQ is supplied as another layer or in solution.Typically, the modified PQQGDH of the present invention is immobilizedon the electrode using glutaraldehyde, then free functional moieties ofglutaraldehyde are blocked by treatment with a reagent having aminegroups.

Measurement of glucose concentration is carried out as described below.Buffer, PQQ, CaCl₂, and a mediator are placed into aconstant-temperature cell and are kept at a constant temperature.Potassium ferricyanide and phenazine methosulfate may be used as amediator. An electrode in which the modified PQQGDH of the presentinvention is immobilized are used as a working electrode, together witha counter electrode (e.g., platinum) and a reference electrode (e.g.,Ag/AgCl electrode). A constant voltage is applied to the carbonelectrode. After the current reaches a constant value, aglucose-containing sample is added and the increase in the current ismeasured. The glucose concentration in the sample can be calculatedusing a calibration curve generated by standard concentration glucosesolutions.

All patents and references cited in this specification are incorporatedby reference. All the contents disclosed in the specifications anddrawings of Japanese Patent Application Nos. 2003-71760 and 2002-196177,on which the application claims priority, are incorporated herein byreference.

The working examples described below further illustrate the inventionwithout limiting the present invention.

EXAMPLE 1

Construction of gene encoding modified PQQGDH enzyme

Mutagenesis was carried out based on the structural gene of PQQGDHderived from Acinetobacter calcoaceticus (SEQ ID NO:2). pGB2 plasmid wasconstructed by inserting the structural gene of PQQGDH derived fromAcinetobacter calcoaceticus into the multi-cloning site of pTrc99Avector (Pharmacia) (FIG.1). The nucleotide sequence encoding glutamineat position 168 or leucine at position 169 of the amino acid sequencedefined SEQ ID NO: 1 was replaced with the nucleotide sequence encodingalanine, glycine, methionine, tryptophan or lysine by standard method ofsite-directed mutagenesis. Also the nucleotide sequence encodingaspartate at position 143 and asparagine at position 428 of the aminoacid sequence defined in SEO ID NO: 1 was replaced with the nucleotidesequence encoding glutamic acid and glycine, respectively. Site specificmutagenesis was performed using the pGB2 plasmid as shown in Fig.2. Thesequences of synthetic oligonucleotide target primers used formutagenesis are shown in Table 1. In order to construct a mutantcontaining two mutations, two oligonucleotide target primers were usedsimultaneously for mutagenesis.

TABLE 1 Gln168Ala 5′-ata agc aag cgg gtt acg ccc-3′ Gln168Gly 5′-caa ataagc aag ccc gtt acg ccc ttg-3′ Gln168Leu 5′-caa ata agc aag cag gtt acgccc ttg-3′ Gln168Phe 5′-caa ata agc aag aaa gtt acg ccc ttg-3′ Gln168Ser5′-caa ata agc aag gct gtt acg ccc ttg-3′ Gln168Asn 5′-caa ata agc aaggtt gtt acg ccc ttg-3′ Gln168Asp 5′-caa ata agc aag atc gtt acg cccttg-3′ Gln168Glu 5′-caa ata agc aag ttc gtt acg ccc ttg-3′ Gln168Lys5′-caa ata agc aag ttt gtt acg ccc ttg-3′ Leu169Ala 5′-caa ata agc agcctg gtt acg-3′ Leu169Gly 5′-gaa caa ata agc acc ctg gtt acg ccc-3′Leu169Met 5′-gaa caa ata agc cat ctg gtt acg ccc-3′ Leu169Trp 5′-gaa caaata agc ttt ctg gtt acg ccc-3′ Leu169Lys 5′-gaa caa ata agc cca ctg gttacg ccc-3′ Asp143Glu 5′-cc tga ctg atg ttc ttt tga tga agg-3′ Asn428Thr5′-c atc ttt ttg gac agt tcc ggc agt at-3′

Table 1 shows SEQ ID NOS: 4, 5, 10-16, 6, 7, 17-19, 8, and 9respectively

A template was prepared by inserting the KpnI-HindIII fragmentcontaining part of the gene encoding PQQGDH derived from Acinetobactercalcoaceticus into pKF18k vector plasmid (TaKaRa). A mixture of template(50 fmol), selection primer (5 pmol) supplied in Mutan-Express Km kit,phosphorylated target primer (50 pmol), and the annealing buffersupplied in the kit ( 1/10 of total volume (20 μl)) was prepared, andplasmid DNA was denatured to single-strand by heating at 100° C. for 3minutes. The selection primer was designed for the reversion ofdouble-amber mutation on the Kanamycin resistance gene of the pKF18kplasmid. Plasmid DNA was put on ice for 5 minutes for annealing of theprimers. A complementary strand was synthesized by adding the followingreagents: 3 μl of extension buffer supplied in the kit, 1 μl of T4 DNAligase, 1 μl of T4 DNA polymerase, and 5 μl of sterilized water. E. coliBMH71-18mutS, a DNA mismatch repair deficient strain, was transformedwith the synthesized DNA and cultured overnight with vigorous shaking toamplify the plasmid.

Then, the plasmid was extracted from the bacteria and transformed intoE. coli MV1184, and the plasmid was extracted from the colonies. Thesequence of the plasmid was determined to confirm successfulintroduction of the desired mutations. Kpn I-Hind III gene fragmentencoding wild type PQQGDH on pGB2 plasmid was replaced with the fragmentcontaining the mutation to construct a series of mutated PQQGDH genes.

EXAMPLE 2

Preparation of Modified Enzyme

A gene encoding wild type or modified PQQGDH was inserted into themulti-cloning site of pTrc99A (Pharmacia), and the constructed plasmidwas transformed into E. coli DH5α. Transformants were cultured in 450 mlof L-broth containing 50 μg/ml of ampicillin and 30 μg/ml ofchloramphenicol using a Sakaguchi flask at 37° C. with vigorous shaking,and then inoculated in 7 L of L-broth containing 1 mM CaCl₂ and 500 μMPQQ. After three hours of cultivation, IPTG was added to a finalconcentration of 0.3 mM, and cultivation was continued for another 1.5hours. The cells were collected by centrifugation (5000×g, 10 min, 4°C.) and washed with 0.85% NaCl twice. The cells were crushed with Frenchpress (110 MPa), and centrifuged twice (10000×g, 15 min, 4° C.) toremove the debris. The supernatant was ultracentrifuged (160,500×g(40,000 rpm), 90 min, 4° C.) to obtain a water-soluble fraction. Thisfraction was used in the subsequent experiments as a crude enzymepreparation.

EXAMPLE 3

Measurement of Enzyme Activity

Each of the crude enzyme preparation of wild type PQQGDH and modifiedPQQGDHs obtained in Example 2 was converted to a holoenzyme in thepresence of 1 μM PQQ and 1 mM CaCl₂ for 1 hour or more. The solution wasdivided into aliquots of 187 μl each, and mixed with 3 μl of activationreagents (6 mM DCIPA 48 μl, 600 mM PMS 8 μl, 10 mM phosphate buffer pH7.0 16 μl) and 10 μl of D-glucose of various concentrations to measurethe enzyme activity.

Enzyme activity was measured in MOPS-NaOH buffer (pH7.0) containing PMS(phenazine methosulfate)-DCIP (2, 6-dichlorophenolindophenol). Changesin absorbance of DCIP was recorded with a spectrophotometer at 600 nm,and the reduction rate of absorbance was defined as the reaction rate ofthe enzyme. In this measurement, enzyme activity which reduced 1 μmol ofDCIP in one minute was defined as 1 unit. The molar absorptioncoefficient of DCIP at pH 7.0 was 16.3 mM⁻¹.

Km was calculated from the plots of substrate concentration vs enzymeactivity. The results are shown in Table 2.

TABLE 2 Km value for glucose(mM) Vmax (U/mg) Wild type 30 129 Gln168Ala50 123 Gln168Gly 36 94 Leu169Ala 177 42 Leu169Gly 157 46 Leu169Met 98176 Leu169Trp 25 17 Leu169Lys 41 36

EXAMPLE 4

Evaluation of Substrate Specificity

Substrate specificity of the crude preparation of the modified enzymeswas examined. Each of the crude enzyme preparation of wild type PQQGDHand modified PQQGDHs obtained in Example 2 was converted to a holoenzymein the presence of 1 μM PQQ and 1 mM CaCl₂ for 1 hour or more. Thesolution was divided into aliquots of 187 μl each, and mixed with 3 μlof activation reagents (6 mM DCIPA 48 μl, 600 mM PMS 8 μl, 10 mMphosphate buffer pH 7.0 16 μl) and substrate. Solution of glucose orother sugars (400 mM) was added as a substrate to a final concentrationof 20 mM or 100 mM, and the mixture was incubated for 30 minutes at roomtemperature. The enzyme activity was measured in the same manner asExample 3. The values were calculated as relative activity to glucose(100%). The results are shown in Table 3-6.

TABLE 3 Wild type Gln168Ala Gln168Gly Leu169Ala Leu169Gly Substrateconc. 20 mM 20 mM 20 mM 20 mM 20 mM Glucose 100 (%) 100 (%) 100 (%) 100(%) 100 (%) Allose 45 29 34 50 39 3-O-m-glucose 82 80 101 66 60Galactose 8 10 12 34 26 Maltose 49 20 24 39 30 Lactose 53 56 40 64 56Cellobiose 85 138 85 84 71

TABLE 4 Wild type Gln168Ala Gln168Gly Leu169Ala Leu169Gly Substrateconc. 100 mM 100 mM 100 mM 100 mM 100 mM Glucose 100 (%) 100 (%) 100 (%)100 (%) 100 (%) Allose 62 41 45 47 35 3-O-m-glucose 92 93 98 86 59Galactose 8 6 19 25 17 Maltose 51 56 44 50 46 Lactose 51 56 44 50 46Cellobiose 42 73 59 59 39

TABLE 5 Wild type Gln168Leu Gln168Phe Gln168Ser Gln168Asp Gln168GluSubstrate conc. 20 mM 100 mM 20 mM 100 mM 20 mM 20 mM 20 mM 100 mM 20 mMGlucose 100 (%) 100 (%) 100 (%) 100 (%) 100 (%) 100 (%) 100 (%) 100 (%)100 (%) 2-Deoxyglucose 0 5 0 0 0 2 0 0 0 mannose 7 9 0 0 2 5 0 0 0allose 41 65 70 78 31 41 26 17 16 3-O-m-glucose 80 97 84 90 62 89 46 5063 galactose 8 6 23 20 61 19 1 2 4 xylose 5 8 17 26 28 7 0 0 0 lactose58 59 63 54 105 66 57 53 60 maltose 67 55 55 36 31 35 8 1 8 cellobiose85 44 90 55 — — 148 82 —

TABLE 6 Wild type Leu169Met Leu169Trp Leu169Lys Substrate 20 mM 20 mM 20mM 20 mM conc. Glucose 100 (%) 100 (%) 100 (%) 100 (%) Galactose 11 3624 43 Xylose 7 17 6 8 Lactose 61 59 76 48 Maltose 61 39 17 31

In addition, the enzyme activity of the modified enzyme of the presentinvention carrying a double mutation was measured. The results are shownin Table 7, 8. Each modified enzyme of the present invention showedhigher reactivity to glucose than to maltose.

TABLE 7 Asp143Glu/ Gln168Gly/ Asn428Thr Asn428Thr Substrate conc. 20 mM20 mM Glucose 100 (%) 100 (%) Allose 2 32 3-O-m-glucose 4 98 Galactose 214 Maltose 2 46 Lactose 12 21

TABLE 8 Asp143Glu/ Asp143Glu/ Asp143Glu/ Wild type Leu168Ala Leu168GlyGln168Leu Substrate conc. 20 mM 100 mM 20 mM 100 mM 20 mM 100 mM 100 mMGlucose 100 (%) 100 (%) 100 (%) 100 (%) 100 (%) 100 (%) 100 (%)2-deoxyglucose 0 5 0 0 0 0 0 Mannose 7 9 1 0 0 0 0 Allose 41 65 6 5 6 72 3-O-m-glucose 80 97 2 4 6 8 2 Galactose 8 6 5 7 1 4 21 Xylose 5 8 0 10 0 5 Lactose 58 59 58 61 50 43 57 Maltose 67 55 11 11 1 5 22 Cellobiose85 44 — 107 215 130 195 Asp143Glu/ Asp143Glu/ Asp143Glu/ Asp143Glu/Gln168Ser Gln168Asn Gln168Glu Gln168Lys Substrate conc. 20 mM 100 mM 20mM 100 mM 20 mM 100 mM Glucose 100 (%) 100 (%) 100 (%) 100 (%) 100 (%)100 (%) 2-deoxyglucose 0 0 2 5 1 8 Mannose 0 0 2 9 3 11 Allose 1 0 16 265 63 3-O-m-glucose 0 0 9 14 6 35 Galactose 3 0 10 12 9 70 Xylose 0 0 3 92 9 Lactose 61 43 49 44 54 90 Maltose 6 3 42 40 16 49 Cellobiose 240 131— 61 — 74

EXAMPLE 5

Dependency on Substrate Concentration

Dependency on substrate concentration of modified enzymes of the presentinvention was examined. Each modified enzyme was converted to aholoenzyme in the presence of 1 μM PQQ and 1 mM CaCl₂ for 1 hour ormore. Following the method for measuring the enzyme activity describedin Example 3, the changes in absorbance of DCIP were measured with aspectrophotometer at 600 nm as an indicator. The results are shown inFIG. 3. The modified PQQGDH of the present invention was saturated athigher concentration of glucose compared with the wild type. For bothmodified and wild type PQQGDHs, substrate inhibition was not observed upto the glucose concentration of 200 mM, and Ksi was observed to be 200mM or more.

EXAMPLE 6

Purification of Enzyme

Each of the crude enzyme preparation of wild type and Gln192Asp obtainedin Example 2 was adsorbed in a cation exchange chromatography columnfilled with TSKgel CM-TOYOPEARL 650M (Toso Co.) and equilibrated with 10mM phosphate buffer, pH 7.0. The column was washed with 750 ml of 10 mMphosphate buffer, pH 7.0 and the enzyme was eluted with 10 mM phosphatebuffer, pH 7.0 containing from 0 M to 0.2 M NaCl. The flow rate was 5mL/min. Fractions showing GDH activity were collected and dialyzedagainst 10 mM MOPS-NaOH buffer (pH 7.0) overnight. In this manner,modified PQQGDH protein was purified which exhibited a single band underelectrophoresis. Enzyme activity for various substrates of the purifiedenzyme was measured. The results are shown in Tables 9-10.

TABLE 9 wild type Glu168Asp Km Vmax kcat kcat/Km Km Vmax kcat kcat/Km(mM) (U/mg) (sec⁻¹) (mM⁻¹ · sec⁻¹) (mM) (U/mg) (sec⁻¹) (mM⁻¹ · sec⁻¹)glucose 25.0 4610 3860 154 (100%) 50.0 475 398  8.0 (100%) allose 35.52997 2509 71 (46%) 57.2 226 189 3.3 (42%) 3-O-m-glucose 28.7 3596 3011105 (68%) 64.4 310 260 4.0 (51%) galactose 5.3 277 232 44 (29%) 118.9137 115 1.0 (12%) lactose 18.9 1982 1659 88 (57%) 75.0 390 327 4.4 (54%)maltose 26.0 2305 1930 74 (48%) 95.8 77 64 0.7 (8%)

TABLE 10 Asp143Glu/Asn428Thr Km kcat kcat/Km (mM) (sec⁻¹) (mM⁻¹ · sec⁻¹)Glucose 48 1193 25 (100%) Allose 182 73 0.4 (2%) 3-O-m-glucose 198 2151.1 (4%) Galactose 145 89 0.6 (2%) Lactose 55 167 3 (12%) Maltose 147 650.4 (2%) Cellobiose 16 226 14 (56%)

EXAMPLE 7

Preparation of Enzyme Sensor and its Evaluation

Twenty mg of carbon paste was added to 5 units of the modified enzymeGln168Ala and freeze-dried. The mixture was applied on the surface of acarbon paste electrode filled with approximately 40 mg of carbon paste,and the electrode was polished on a filter paper. This electrode wastreated with MOPS buffer (pH 7.0) containing 1% glutaraldehyde for 30minutes at room temperature and then treated with MOPS buffer (pH 7.0)containing 20 mM lysine for 20 minutes at room temperature to blockunreacted glutaraldehyde. The electrode was equilibrated in 10 mM MOPSbuffer (pH 7.0) for one hour or more at room temperature, then stored at4° C.

The glucose concentration was measured using the glucose sensor thusprepared. Glucose concentration was quantified in the range from 0.1 mMto 5 mM by using the glucose sensor prepared with the modified PQQGDH ofthe invention.

INDUSTRIAL APPLICABILITY

The modified water-soluble PQQGDH of the present invention has highglucose selectivity, thus is useful as a device of a sensor formeasuring blood glucose level.

1. An isolated mutant water-soluble glucose dehydrogenase havingpyrroloquinoline quinone as a coenzyme, wherein said mutant is a mutantof a glucose dehydrogenase comprising the amino acid sequence of SEQ IDNO: 1, and wherein said mutant consists of an amino acid substitutionselected from the group consisting of: (1) glutamine at position 168 ofSEQ ID NO:1 is substituted with glycine, glutamic acid, leucine,phenylalanine, serine or aspartic acid, optionally combined with (a) asubstitution wherein aspartate at position 143 of SEQ ID NO: 1 issubstituted with glutamic acid or (b) a substitution wherein asparagineat position 428 of SEQ ID NO:1 is substituted with threonine; (2)leucine at position 169 of SEQ ID NO:1 substituted with alanine,glycine, methionine, tryptophan or lysine, optionally combined with (a)a substitution wherein aspartate at position 143 of SEQ ID NO:1 issubstituted with glutamic acid or (b) a substitution wherein asparagineat position 428 of SEQ ID NO:1 is substituted with threonine ; and (3)aspartate at position 143 of SEQ ID NO:1 is substituted with glutamicacid and asparagine at position 428 of SEQ ID NO: 1 is substituted withthreonine.
 2. A glucose assay kit comprising the modified glucosedehydrogenase as claimed in claim
 1. 3. A glucose sensor comprising themodified glucose dehydrogenase as claimed in claim
 1. 4. The mutantglucose dehydrogenase as claimed in claim 1, wherein glutamine atposition 168 of SEQ ID NO:1 is substituted with glycine, glutamic acid,leucine, phenylalanine, serine or aspartic acid.
 5. The mutant glucosedehydrogenase as claimed in claim 1, wherein leucine at position 169 ofSEQ ID NO:1 is substituted wit alanine, glycine, methionine, tryptophanor lysine.
 6. The mutant glucose dehydrogenase as claimed in claim 1,wherein aspartate at position 143 of SEQ ID NO:1 is substituted withglutamic acid, and asparagine at position 428 of SEQ ID NO:1 issubstituted with threonine.
 7. The mutant glucose dehydrogenase asclaimed in claim 1, wherein glutamine at position 168 of SEQ ID NO:1 issubstituted with glycine, glutamic acid, leucine, phenylalanine, serineor aspartic acid in SEQ ID NO:1, and aspartate at position 143 of SEQ IDNO:1 is substituted wit glutamic acid.
 8. The mutant glucosedehydrogenase as claimed in claim 1, wherein glutamine at position 168of SEQ ID NO:1 is substituted with glycine, glutamic acid, leucine,phenylalanine, serine or aspartic acid in SEQ ID NO:1, and asparagine at428 of SEQ ID NO:1 is substituted with threonine.
 9. The mutant glucosedehydrogenase as claimed in claim 1, wherein leucine at position 169 ofSEQ ID NO:1 is substituted with alanine, glycine, methionine, tryptophanor lysine and aspartate at position 143 of SEQ ID NO:1 is substitutedwit glutamic acid.
 10. The mutant glucose dehydrogenase as claimed inclaim 1, wherein leucine at 169 of SEQ ID NO:1 is substituted withalanine, glycine, methionine, tryptophan or lysine and asparagine atposition 428 of SEQ ID NO:1 is substituted with threonine.
 11. Anisolated mutant water-soluble glucose dehydrogenase havingpyrroloquinoline quinone as a coenzyme, wherein said mutant is a mutantof a glucose dehydrogenase comprising the amino acid sequence of SEQ IDNO:1, and wherein said mutant consist of an amino acid substitutionwherein glutamine at position 168 of SEQ ID NO:1 is substituted withglycine, glutamic acid, leucine, phenylalanine, serine or aspartic acid.